The complement system is responsible for initiating and amplifying the inflammatory response to microbial infection and other acute insults. Inappropriate activation of complement has been implicated in pathological situations. For instance, the complement system has been implicated in contributing to the pathogenesis of several acute and chronic conditions, including atherosclerosis, ischemia-reperfusion following acute myocardial infarction, Henoch-Schonlein purpura nephritis, immune complex vasculitis, rheumatoid arthritis, arteritis, aneurysm, stroke, cardiomyopathy, hemorrhagic shock, crush injury, multiple organ failure, hypovolemic shock and intestinal ischemia, transplant rejection, cardiac Surgery, PTCA, spontaneous abortion, neuronal injury, spinal cord injury, myasthenia gravis, Huntington's disease, amyotrophic lateral sclerosis, multiple sclerosis, Guillain Barre syndrome, Parkinson's disease, Alzheimer's disease, acute respiratory distress syndrome, asthma, chronic obstructive pulmonary disease, transfusion-related acute lung injury, acute lung injury, Goodpasture's disease, myocardial infarction, post-cardiopulmonary bypass inflammation, cardiopulmonary bypass, septic shock, transplant rejection, xeno transplantation, burn injury, systemic lupus erythematosus, membranous nephritis, Berger's disease, psoriasis, pemphigoid, dermatomyositis, anti-phospholipid syndrome, inflammatory bowel disease, hemodialysis, leukopheresis, plasmapheresis, heparin-induced extracorporeal membrane oxygenation LDL precipitation, extracorporeal membrane oxygenation, and macular degeneration.
Complement can be activated through three distinct enzymatic cascades, referred to as the “classical”, “Lectin/MBL”, and “alternative” pathways (CP, MBL, and AP respectively). These pathways are shown schematically in FIG. 1. The AP is responsible for 80-95% of total complement activity. Both classical and alternative pathways have the ability to produce C3a and C5a. However, the level of these anaphylatoxins varies depending upon which pathway is active. Lectin pathway is a variation of the classical pathway. Alternative pathway is activated in a number of disease indications. There are three specific proteins Factors B, D, and P that play a major role in the initiation and propagation of the AP. The terminal complex is known as MAC which is responsible for lysis. Both C3a and C5a are potent anaphylatoxins that are responsible for activating platelets, neutrophils, and monocytes. As a result, inflammatory molecules such as elastase, TNF, IL-1, VEGF, and peroxides are released.
FIG. 2 illustrates a schematic of AP activation. As a result of C3 tick over, C3b is generated. In the schematic, assumption has been made that tick-over of C3 and cleavage of C3 generates the same activated C3b with the released C3a. Activated C3b binds properdin oligomers present in blood to generated (P)n (C3b)n complex. Factor B having higher affinity to properdin bound C3b makes the complex PC3bB, which is then cleaved by factor D to generate PC3bBb. This active convertase cleaves additional C3 to make C3b and release C3a. The same C3 convertase with additional C3b molecules forms C5 convertase. The C5 convertase or C3 convertase cleave C5 to make C5b and C5a. The C5b molecule inserts into the lipid bilayer and forms the nucleus for MAC deposition.
Factor B is composed of two discrete domains Ba (molecular weight, 33 kDa) and Bb (molecular weight, 60 kDa). The Ba domain consists of three short consensus repeats known as SCR1, SCR2, and SCR3 (FIG. 3). It has been shown, using mutation analysis and with the use of specific Ba monoclonal antibodies that the factor B functional domain is located in the SCR3 region. Such region was used to produce antibodies that demonstrated clinical benefit in several animal models of diseases. The Bb domain of factor B contains the Von Willowbrand (VWF) domain in addition to the serine protease domain. It is clear from various studies that it is the Ba domain that is important for factor B function, and that inhibition of factor B binding to C3b was required for inhibition of complement activity.
Properdin, a small but important molecule binds C3b to form P-C3b complex and such binding is high affinity. Factor B binds both free C3b and P-C3 to form C3bB and PC3bB complexes. These complexes are cleaved by factor D to form, C3bBb and PC3bBb, both of which possess C3-convertase activity. The resulting convertase can cleave C3, into C3b and C3a. The newly produced C3b fragment, which covalently attaches to the target and then interacts with factors B and D to form the additional alternative pathway C3 convertase molecules.
It is known that the alternative pathway C3-convertase is stabilized by C3b-bound properdin. Since the substrate for the alternative pathway C3-convertase is C3, C3 is therefore both a component and a product of the reaction. As the C3-convertase generates increasing amounts of C3b, an amplification loop is established. Furthermore, the classical pathway can also generate C3b which can bind factor B and thereby engage the alternative pathway even though the trigger is CP mediated. This allows more C3b to deposit on a target leading to enhanced amplification of AP activation. All three, the classical, the lectin, and the alternative pathways converge at C3, which is cleaved by the C3 convertase to form C3b and C3a. C3a is a potent anaphylatoxin and has been implicated in the pathogenesis of a variety of clinical indications. C3a activates neutrophils, monocytes, platelets, mastcells, and T lymphocytes. C3a has been shown to be important for the induction of paw edema in an adjuvant-induced arthritis model.
Addition of newly formed C3b to the existing C3 convertase forms C5 convertase, which cleaves C5 to produce C5b and C5a. C5a similar to C3a is also a potent anaphylatoxin that causes alterations in smooth muscle, in vascular tone, and in vascular permeability. It is also a powerful chemotaxin and an activator of neutrophils, monocytes, platelets, endothelial cells, and T lymphocytes. C5a-mediated cellular activation can significantly amplify inflammatory responses by inducing the release of additional inflammatory mediators, including cytokines, hydrolytic enzymes, arachadonic acid metabolites and reactive oxygen species.
The cleavage of C5 produces C5b and C5a. Anaphylatoxin C5a is released and C5b inserts itself into the lipid bilayer and acts as a nucleus for C6, C7, C8, and C9 deposition to form the C5b-9 complex at the surface of the target cell. C5b-9 is also known as the membrane attack complex (MAC). There is now strong evidence that MAC may play an important role in inflammation in addition to its role as a lytic pore-forming complex. In addition to the proven role of C3a, C5a in platelet activation, C5b-9 is also known to mediate activation of platelets. Thus, there is significant evidence suggesting C3a, C5a, and MAC involvement in activation of platelets. Regardless of the method of platelet activation, activated platelets express CD62P also called P-selectin. P-selectin also mediates platelet-monocyte conjugation, and such binding triggers the release of tissue factor from monocytes. One result of such conjugate formation is the removal of platelets from the circulation, a phenomenon that can contribute to the development of thrombocytopenia.
While complement activation provides a valuable first-line defense against potential pathogens, the activities of complement that promote a protective inflammatory response can also represent a potential threat to the host. For example, C3a and C5a anaphylatoxins recruit and activate neutrophils, monocytes and platelets to the pathological site. These activated cells are indiscriminate in their release of destructive enzymes and may cause organ damage. Currently, there are no approved drugs exist that can inhibit the damages caused by the inappropriate activation of the complement pathway. Based upon the available clinical data, it appears that in most acute injury settings, complement activation is mediated predominantly via the alternative pathway. Therefore, developing suitable methods that inhibit only this pathway without completely obviating the immune defense capabilities would be highly desirable. This would leave the classical pathway intact to handle immune complex processing and to aid in host defense against infection.
Factor B plays a key role in the amplification loop of the alternative pathway since it provides the catalytic subunit, Bb, for the C3-convertase (PC3bBb). Since factor B is an essential component of the alternative pathway, it presents an attractive target for specifically inhibiting this pathway. Factor B by itself is a zymogen with no known catalytic activity, but after binding to PC3b complex, factor B is cleaved by factor D to release Ba. It has been shown that factor B binds C3b through regions found within each of the Ba and Bb subunits. Inhibitors of factor Bb binders should results in selective inhibition of factor B function thereby preventing formation of C3a, C5a and C5b-9, which are responsible for many deleterious effects mentioned previously. Based on the results described in this patent application, it should be possible to develop anti-Bb specific inhibitors or inhibition methods that (a) will prevent factor B function by blocking PC3bBb activity and/or (b) suppress factor B cleavage that prevent Bb generation. These inhibitors appear to be inactivators of the C3 convertase enzymatic activity without disrupting the factor B interaction with C3b. We have evaluated the inhibitory activity of the anti-factor Bb antibodies for their potential role in blocking the AP activation. These antibodies prevent factor B function both in vitro and in whole blood. Other anti-factor Ba monoclonal antibodies have also been developed and tested in animal models of disease. These antibodies prevent factor B binding to C3b and hence block the activation of the complement cascade.